title: A system for viably maintaining a stirred suspension of multicellular spheroids during NMR spectroscopy.
abstract: We have developed a system for the perfusion of a stirred suspension of multicellular spheroids during nuclear magnetic resonance spectroscopy. Measurement of the medium temperature, pH, oxygen tension, and glucose and lactate concentrations demonstrated that the macroenvironmental conditions around the spheroids during perfusion matched those in standard spinner culture flasks. Spheroids cultured in the NMR perfusion chamber for up to 48 h were virtually identical to spheroids cultured under standard conditions in terms of volume and cell number growth, the extent of central necrosis, cellular clonogenicity, and proliferative status. To avoid problems in interpreting the NMR spectra, we have used a medium containing 10% of the normal inorganic phosphate concentration; comparative growth and NMR studies showed that this medium had no effect on the results reported. 31P NMR spectroscopic analysis demonstrated that the mean pH, nucleotide triphosphate (NTP) to inorganic phosphate (Pi) ratio, the total amount of NTP, and the total energy charge were essentially constant over 8 h of analysis. Stopping the stirring of the spheroid culture during analysis resulted in depletion of the nucleotide phosphate pool in 30 min, with an accumulation of Pi and a shift to a more acid intracellular pH. This effect could be reversed if stirring was resumed within 30 min. Stopping the perfusion while maintaining stirring resulted in a deterioration of the 31P spectra until no high energy phosphates remained at 120 min and the pH fell to approximately 6. This effect was also partially reversible after 30 min of reperfusion, with recovery to a normal 31P spectrum requiring 10 h. The combination of the spheroid model system with 31P NMR spectroscopic analysis will provide a powerful tool for investigating basic questions about the regulation of tumor cell energy metabolism and viability.
authors.affiliation: Life Sciences Division, Los Alamos National Laboratory, University of California, NM 87545.
authors.forename: J P
authors.initials: JP
authors.lastname: Freyer
authors.forename: N H
authors.initials: NH
authors.lastname: Fink
authors.forename: P L
authors.initials: PL
authors.lastname: Schor
authors.forename: J R
authors.initials: JR
authors.lastname: Coulter
authors.forename: M
authors.initials: M
authors.lastname: Neeman
authors.forename: L O
authors.initials: LO
authors.lastname: Sillerud
mesh_terms: D000818:Animals; D002470:Cell Survival / Q000502:physiology; D003470:Culture Media; D004734:Energy Metabolism / Q000502:physiology; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008954:Models, Biological; D010477:Perfusion / Q000379:methods*; D010761:Phosphorus Radioisotopes; D013535:Suspensions; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000502:physiology*
chemical_list: D003470:Culture Media; D010761:Phosphorus Radioisotopes; D013535:Suspensions